Proteomics methods provide a non-biased way of detecting and quantitating proteins and peptides, without prior knowledge of the particular protein or peptide. However, typical proteomics schemes detect only the most abundant proteins in a given tissue. While changes in these abundant molecules can be important in understanding the consequence of drug abuse, it is also important to examine the less abundant molecules such as those involved in signal transmission between cells. We have developed a novel method to selectively isolate peptide processing intermediates from fat/fat mouse brain, and have modified this method to permit the quantitation of relative levels of the peptides in two different groups of animals by differential isotopic labeling. The peptides isolated by this technique are substrates of carboxypeptidase E, an important enzyme in the biosynthesis of numerous neuroendocrine peptides. In Aim 1, we will use our quantitative peptidomics method to examine the effect of cocaine or methamphetarnine administration on the relative levels of peptides in specific brain areas such as the hippocampus, striatum, prefrontal cortex, and hypothalamus. Aim 2 will test whether any peptides found to be altered by the chronic cocaine or methamphetamine in the fat mice are similarly altered in wild type mice. Because the peptidomics technique cannot detect neuropeptides in wild type mice due to the high background from other molecules, this Aim will use traditional techniques such as radioimmunoassay and Northern blot analysis to examine peptide and mRNA levels. Taken together, these studies will provide a better understanding of the regulation of neuropeptides, both known and novel, in drug abuse.